Inside-Out Radial Migration Facilitates Lineage-Dependent Neocortical Microcircuit Assembly  Shuijin He, Zhizhong Li, Shaoyu Ge, Yong-Chun Yu, Song-Hai Shi  Neuron  Volume 86, Issue 5, Pages 1159-1166 (June 2015) DOI: 10.1016/j.neuron.2015.05.002 Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Preferential Formation of Strong Electrical Coupling between RGP and Its Progeny (A) Representative images of an EGFP-expressing RGP in the VZ (cell 1, arrow) and its progeny in the SVZ (cell 3) (green) and a nearby non-EGFP-expressing RGP in the VZ (cell 2) and a nearby non-EGFP-expressing SVZ cell (cell 4) on the same side in the E16 neocortex. Cells were subjected to quadruple whole-cell recording, filled with Alexa Fluor 568 hydrazide (red), and stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 30 μm. (B) Example current traces of the four cells evoked by 10 mV step changes in membrane potential from +10 mV to +50 mV. Note that inward voltage-dependent sodium-channel-mediated currents are elicited in cells 3 and 4 (arrows), but not in cells 1 and 2. Scale bars: 50 pA, 5 ms. (C) Example traces of currents elicited by sequential depolarization of one of the four cells from −70 mV to +30 mV. Green circles indicate an EGFP-expressing RGP and its progeny in the SVZ, whereas white circles indicate a nearby non-EGFP-expressing RGP and a nearby non-EGFP-expressing SVZ cell. The average traces are shown in corresponding rectangles with the driver cell at the main diagonal. Red arrows and arrowheads indicate detectable junctional currents between RGPs and between RGP and SVZ progeny, respectively. Similar symbols and panel display are used in subsequent figures. Scale bars: 500 pA, 100 ms, and 5 pA. (D) Morphological reconstruction of the four cells. The wavy lines with arrows indicate electrical coupling, and the thickness of the line reflects the coupling strength. (E) Summary of the rate of electrical coupling observed between nearby RGPs, between EGFP-expressing RGPs and their progeny, between non-EGFP-expressing RGPs and SVZ cells adjacent to EGFP-expressing cells on the same side, and between nearby non-sister SVZ cells. (F) Summary of the rate of electrical coupling observed between EGFP-expressing SVZ cells and non-EGFP-expressing RGPs adjacent to EGFP-expressing RGPs and between EGFP-expressing RGPs and non-EGFP-expressing SVZ cells adjacent to EGFP-expressing SVZ cells. (G) Quantification of the coupling conductance observed between EGFP-expressing RGP and progeny pairs and between RGP and non-progeny SVZ cell pairs at E16–E18. Data are presented as mean ± SEM; ∗∗∗, p < 0.001. See also Figure S1. Neuron 2015 86, 1159-1166DOI: (10.1016/j.neuron.2015.05.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Progressive and Preferential Electrical Coupling between Radially Situated Sister Excitatory Neurons in the Embryonic Neocortex (A) Representative images of a pair of EGFP-expressing sister excitatory neurons (green, cells 1 and 2) in the CP of the E18 neocortex subjected to dual whole-cell recording, filled with Alexa Fluor 568 hydrazide (red), and stained with DAPI (blue). Scale bar: 50 μm. (B) Example traces of currents elicited in both cells by sequential depolarization of one of the two cells. Scale bars: 500 pA, 100 ms, and 10 pA. (C) Summary of the rate of electrical coupling between sister neurons at different embryonic stages. (D) Representative images of two radially situated EGFP-expressing sister excitatory neurons (cells 1 and 3, green) and two adjacent non-EGFP-expressing control excitatory neurons on the same side (cells 2 and 4) in the CP of E18 neocortex subjected to quadruple whole-cell recording. Scale bar: 25 μm. (E) Example traces of currents in the four neurons elicited by sequential depolarization of one of the four cells. Green circles indicate two EGFP-expressing sister excitatory neurons, and white circles indicate two adjacent non-EGFP-expressing non-sibling control excitatory neurons. Average currents are shown in corresponding rectangles with the driver cell at the main diagonal. Red arrowheads indicate detectable junctional currents. Similar symbols and panel display are used in subsequent figures. Scale bars: 500 pA, 100 ms, and 5 pA. (F) Summary of the rate of electrical coupling observed between sister excitatory neurons and their adjacent non-sibling excitatory neurons. Neuron 2015 86, 1159-1166DOI: (10.1016/j.neuron.2015.05.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Preferential Electrical Coupling between Sister Excitatory Neurons, but Not between RGP and progeny, Is Abolished in the Neocortex of Reeler Mice (A–C) Dual whole-cell recording of an EGFP-expressing RGP (cell 1, green, [A]) and its neuronal progeny in the SVZ (cell 2, green, [A]) with obvious sodium currents (arrow, [B]) in the embryonic Reeler neocortex. Depolarization of one cell evoked currents simultaneously in both cells (C), indicating electrical coupling between the RGP and its neuronal progeny. Scale bars: 20 μm (A); 100 pA and 10 ms (B); 200 pA, 100 ms, and 4 pA (C). (D) Summary of the rate of electrical coupling between RGPs and their neuronal progeny in the SVZ of the wild type (WT) or the Reeler neocortex. (E–H) Quadruple whole-cell recordings of two EGFP-expressing sister excitatory neurons (1 and 3, green) and two adjacent non-EGFP-expressing control excitatory neurons (2 and 4) in the embryonic and neonatal WT ([E] and [F]) and Reeler ([G] and [H]) neocortices. Scale bars: 25 μm ([E] and [G]); 250 pA, 100 ms, 10 pA ([F] and [H]). (I and J) Summary of the rate of electrical coupling observed between sister excitatory neurons and their adjacent non-sister excitatory neurons in the WT and Reeler neocortices at E17 and E18 (I) and P1–P4 (J). ∗, p < 0.05; ∗∗∗, p < 0.001. See also Figures S2 and S3. Neuron 2015 86, 1159-1166DOI: (10.1016/j.neuron.2015.05.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Preferential Electrical Coupling between Sister Excitatory Neurons Is Disrupted by Clonal Knockdown of DAB1 (A–D) Quadruple whole-cell recordings of two sister excitatory neurons expressing EGFP/Vector alone ([A] and [B]) or EGFP/Dab1 shRNA-3 ([C] and [D]) (1 and 3, green) and two adjacent non-EGFP-expressing control excitatory neurons (2 and 4) in the neonatal neocortex. Scale bars: 20 μm ([A] and [C]); 500 pA, 100 ms, and 5 pA ([B] and [D]). (E) Summary of the rate of electrical coupling observed between sister excitatory neurons expressing vector alone, Dab1 shRNA-1, or shRNA-3 and their adjacent non-sister excitatory neurons in the neocortex. ∗∗∗, p < 0.001. See also Figure S4. Neuron 2015 86, 1159-1166DOI: (10.1016/j.neuron.2015.05.002) Copyright © 2015 Elsevier Inc. Terms and Conditions